Here's another way to deal with this nasty problem: don't cover the gel
with buffer initially. Just add enough buffer to cover the ends of the
gel, load your samples and run the gel for a half hour or so. By then
the DNA is into the gel, so you can cover it with buffer. This method
has the added advantage that you can load more volume into each well. I
ALWAYS do this with precious samples.
******************** HAVE GENES, WILL TRAVEL ********************
Joe Bagshaw, Worcester Polytechnic Institute
jbagshaw at wpi.wpi.edu
Roadkill on the information superhighway.
On Fri, 25 Aug 1995, Ole Skovgaard wrote:
> In article <ketilt.4.000D5FD6 at dmf.unit.no> ketilt at dmf.unit.no (Ketil Thorstensen) writes:
> >From: ketilt at dmf.unit.no (Ketil Thorstensen)
> >Subject: DNA "refusing" to be run in agarose gel
> >Date: Fri, 25 Aug 1995 13:22:22
>> >Honored contributors to the molbio.methds-reagnts!
>> >I have a peculiar problem with persuading some of my DNA samples to run in
> >agarose gels.
>> >When loading DNA samples on agarose gels I have on several occasions observed
> >a strange (to me) phenomenon: after application of a sample into a well a
> >streak of the sample may follow the pipette tip upon withdrawal. When the
> >sample in this way comes in contact with the buffer surface, the sample very
> >rapidly spreads out to cover the entire surface. During this process most of
> >the sample in the well is "sucked" out.
> >There is no recognizable pattern as to what sample this may happen with. It
> >is, however, frustrating when precious samples are lost (the last one was a
> >1.9 kb DNA probe) :í-(
> >Could anyone enlighten me as to what I am observing, and how to prevent
> >this from happening again?
>> >Thank you very much,
>> >Ketil Thorstensen, Ph.D.
> >Dept. Clinical Chemistry
> >University Hospital
> >Trondheim, Norway
> >
> It is a well known phenomenon. It is due to the presence of either detergent
> or organic solvent i.e. ethanol/isopropanol from the last precipition before.
> Do your precipitation but remove ALL - repeat ALL - liquid before
> continuation. Do it with add of a pipette, centrifuge and remove the remaining
> liquid. Do not do it by inverting the tube or other silly things that
> biochemists have taught us (accept my apology for being root). Do not dry the
> pellet. Do not wash the pellet. That makes it go away - just remove all
> liquid!! and redisolve in whatever buffer. If there has been phenol in the
> tube change it immediately. Quite an amount of phenol may hide in the tube
> wall.
> Add enough loading buffer for the
> gel to increase density of your sample above that of the gel buffer. Good luck,
> ole
>>> ******************************************
> **** Ole Skovgaard ****
> **** Dept. Life. Sc. & Chemistry ****
> **** Roskilde University, DK ****
> ******************************************
>>